A recently defined family of cytokines, consisting of ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), and interleukin-6 (IL-6), utilize the Jak-Tyk family of cytoplasmic tyrosine kinases. The beta receptor components for this cytokine family, gp130 and LIF receptor beta, constitutively associate with Jak-Tyk kinases. Activation of these kinases occurs as a result of ligand-induced dimerization of the receptor beta components. Unlike other cytokine receptors studied to date, the receptors for the CNTF cytokine family utilize all known members of the Jak-Tyk family, but induce distinct patterns of Jak-Tyk phosphorylation in different cell lines.

Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, interleukin-11, and ciliary neurotrophic factor bind to receptor complexes that share the signal transducer gp130. Upon binding, the ligands rapidly activate DNA binding of acute-phase response factor (APRF), a protein antigenically related to the p91 subunit of the interferon-stimulated gene factor-3 alpha (ISGF-3 alpha). These cytokines caused tyrosine phosphorylation of APRF and ISGF-3 alpha p91. Protein kinases of the Jak family were also rapidly tyrosine phosphorylated, and both APRF and Jak1 associated with gp130. These data indicate that Jak family protein kinases may participate in IL-6 signaling and that APRF may be activated in a complex with gp130.

We have produced a cell line which lacks the protein tyrosine kinase JAK1 and is completely defective in interferon response. Complementation of this mutant with JAK1 restored the response, establishing the requirement for JAK1 in both the interferon-alpha/beta and -gamma signal transduction pathways. The reciprocal interdependence between JAK1 and Tyk2 activities in the interferon-alpha pathway, and between JAK1 and JAK2 in the interferon-gamma pathway, may reflect a requirement for these kinases in the correct assembly of interferon receptor complexes.

tyk2 belongs to the JAK family of nonreceptor protein tyrosine kinases recently found implicated in signaling through a large number of cytokine receptors. These proteins are characterized by a large amino-terminal region and two tandemly arranged kinase domains, a kinase-like and a tyrosine kinase domain. Genetic and biochemical evidence supports the requirement for tyk2 in interferon-alpha/beta binding and signaling. To study the role of the distinct domains of tyk2, constructs lacking one or both kinase domains were stably transfected in recipient cells lacking the endogenous protein. Removal of either or both kinase domains resulted in loss of the in vitro kinase activity. The mutant form truncated of the tyrosine kinase domain was found to reconstitute binding of interferon-alpha 8 and partial signaling. While no contribution of this protein toward interferon-beta binding was evident, increased signaling could be measured. The mutant form lacking both kinase domains did not exhibit any detectable activity. Altogether, these results show that a sequential deletion of domains engenders a sequential loss of function and that the different domains of tyk2 have distinct functions, all essential for full interferon-alpha and -beta binding and signaling.

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-␣ (IFN-␣) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-␣. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-␣ receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-␣. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.The intracellular protein-tyrosine kinases of the Janus kinase (JAK) family play an essential role in cytokine signaling: they interact with receptor components and undergo tyrosine phosphorylation and enzymatic activation upon ligand binding. Their activation is the first step of a cascade of intracellular phosphorylation events ultimately leading to the transcriptional activation of target genes (1, 2). Signaling through the receptor for type I interferons (IFN-␣ and -␤) requires two members of the JAK family, Tyk2 and JAK1 (3, 4). Both enzymes are associated with the receptor, which is composed of IFN-␣ receptor (IFNAR) 1 (5) and IFNAR2-2, the longer splice variant of the IFNAR2 gene (6, 7). Tyk2 was shown to interact with the membrane-proximal region of IFNAR1 (8, 9), and JAK1 with IFNAR2-2 (10, 11). The stoichiometry of the activated receptor͞JAK complex is not known and might differ for the different type I IFNs. Ligand-induced dimerization or oligomerization of the receptor components leads to asymmetric trans-phosphorylation and consequent catalytic activation of Tyk2 and JAK1 (4,12,13).Tyk2 shares with the other members of the JAK family a unique structural framework (see Fig. 1A) comprising seven conserved JAK homology (JH) regions (14). The most Cterminal one (JH1) is a tyrosine kinase (TK) domain, which is flanked by the JH2 or kinase-like (KL) domain of unknown function. The remaining five blocks of homology (JH3 to JH7) extend toward the N terminus of the protein and exhibit variable degrees of conservation among the family members, the most conserved being JH4 with a central core of 18 identical...

BackgroundIn a previous work we showed for the first time that human tumor cells secrete Hsp60 via exosomes, which are considered immunologically active microvesicles involved in tumor progression. This finding raised questions concerning the route followed by Hsp60 to reach the exosomes, its location in them, and whether Hsp60 can be secreted also via other mechanisms, e.g., by the Golgi. We addressed these issues in the work presented here.Principal FindingsWe found that Hsp60 localizes in the tumor cell plasma membrane, is associated with lipid rafts, and ends up in the exosomal membrane. We also found evidence that Hsp60 localizes in the Golgi apparatus and its secretion is prevented by an inhibitor of this organelle.Conclusions/SignificanceWe propose a multistage process for the translocation of Hsp60 from the inside to the outside of the cell that includes a combination of protein traffic pathways and, ultimately, presence of the chaperonin in the circulating blood. The new information presented should help in designing future strategies for research and for developing diagnostic-monitoring means useful in clinical oncology.

We have previously identified a muscle-specific enhancer within the first intron of the human ␤ enolase gene. Present in this enhancer are an A/T-rich box that binds MEF-2 protein(s) and a G-rich box (AGTGGGG-GAGGGGGCTGCG) that interacts with ubiquitously expressed factors. Both elements are required for tissuespecific expression of the gene in skeletal muscle cells. Here, we report the identification and characterization of a Kruppel-like zinc finger protein, termed ␤ enolase repressor factor 1, that binds in a sequence-specific manner to the G-rich box and functions as a repressor of the ␤ enolase gene transcription in transient transfection assays. Using fusion polypeptides of ␤ enolase repressor factor 1 and the yeast GAL4 DNA-binding domain, we have identified an amino-terminal region responsible for the transcriptional repression activity, whereas a carboxyl-terminal region was shown to contain a potential transcriptional activation domain. The expression of this protein decreases in developing skeletal muscles, correlating with lack of binding activity in nuclear extract from adult skeletal tissue, in which novel binding activities have been detected. These results suggest that in addition to the identified factor, which functionally acts as a negative regulator and is enriched in embryonic muscle, the G-rich box binds other factors, presumably exerting a positive control on transcription. The interplay between factors that repress or activate transcription may constitute a developmentally regulated mechanism that modulates ␤ enolase gene expression in skeletal muscle.

In mammals there are at least three isoforms of the glycolytic enzyme enolase encoded by three similar genes:a, p and y. In this report we describe the isolation and characterization of the human a-enolase locus. The gene appears to exist as a single copy in the haploid genome and is composed of 12 exons distributed over more than 18000 bases. The structure of this gene has a high degree of similarity to that of the human and rat y-enolase genes, with identical positions for all the intron regions. Primer extension and S1 nuclease protection experiments indicate that transcription is initiated at multiple sites. The putative promoter region, like that of other housekeeping genes, lacks canonical TATA and CAAT boxes, is extremely G + C-rich and contains several potential SP1 binding sites. Furthermore, various sequences similar to known regulatory elements were detected.The functional role of multiple enzyme isoforms and the regulatory mechanisms required for developmental and tissuespecific expression of these isoforms are still largely unknown. The glycolytic enzyme enolase (2-phospo-~-glycerate hydrolase) represents a suitable model for the study of these mechanisms. Three distinct isoforms of the enzyme, referred to as a or non-neuronal enolase, p or muscle-specific enolase and y or neuron-specific enolase are present in both avian and mammalian tissues [l]. The active enzyme is a homodimer of non-covalently linked subunits, each a, p and y subunit is encoded by a separate gene as previously proposed [2] and recently established by cDNA cloning in human [3-51, rat [6 -81, and mouse [9].While the y isoform is mainly detected in cells of neuronal origin and the p isoform is found in adult skeletal muscle, the a isoform is widely distributed among different tissues and is the major form of enolase present in the early stage of embryonic development. Isoform switch occurs along with terminal differentiation in neurons and skeletal muscle cells from the a-to the y-and p-enolase respectively [2, 10, 111. Although the isolated isoenzymes have been studied for many years and a substantial body of information on their biochemical, kinetic, and immunological properties [I21 as well as on the crystallographic structure [I31 has been collected, very little is known about the control of expression of the enolase genes. Beside the already mentioned differential expression during development and in various cell types, transcriptional induction of the a-enolase gene has been shown upon mitogenic stimulation of human peripheral-blood lymphocytes [3], as well as in quiescent rat fibroblast stimulated with growth factors or serum [14]. High levels of expression of the y isoform have been detected in many tumors of nonneuronal origin [5] suggesting lack of tissue-specific control in transformed cells. Furthermore, the finding that one of the two enolase isoenzymes of the budding yeast Saccharomyces cerevisiue may be involved in both thermal tolerance and growth control [15] and the identification of the lens structural prote...